1. Field of the Invention
The present invention is generally directed to a triple-stranded DNA, a method of forming the same, and Southern hybridization employing the same.
2. Discussion of the Background
Conventional methods are known to form a complex including double-stranded and single-stranded DNAs. That is to say, as shown in FIG. 19, a target DNA (i.e. a double-stranded DNA) and a probe DNA (i.e. a single-stranded DNA) are prepared. The probe DNA (i.e. the single-stranded DNA) has a base sequence which is substantially complementary to a portion of a base sequence of one of DNA-chains of the target DNA. These DNAs and a RecA of Escherichia coli are mixed into a solution which includes a buffer etc and the resultant mixture is held at a predetermined temperature for a sufficient time duration.
Then, a DNA-protein complex is obtained which is made up of the target DNA, the probe DNA, and the RecA protein. In detail, first of all, the RecA protein is bound to the probe DNA to form a probe DNA-RecA protein complex. Subsequently, the resultant complex or the probe DNA-RecA protein complex is bound to the target DNA to form a DNA-protein complex which includes a three-chain formation region. At this stage, the probe DNA is believed to bind to a region of the target DNA which has a base sequence complementary to the probe DNA. The DNA-protein complex at this state, though it has the three-chain formation region, is relatively stable (see B. Jagadeeshwar Rao et al., Proc. Natl. Sci. USA, 88,2984-2988 (1991), Gurucharan Reddy at al., Biochemistry, 33,11486-11492(1994), and Efim I. Golumb eat al., Mutation Research, 351, 117-124 (1996)).
However, as shown in FIG. 19, if the RecA protein is deactivated in such a manner that the DNA-protein complex is mixed with a sodium dodecyl sulfate (SDS) and/or a protein splitting enzyme (e.g. protease K) and the resultant mix is held at a temperature for a sufficient time duration, the bonding between the target DNA and the probe DNA dissociates in addition to a deleting the RecA protein from the DNA-protein complex. That is to say, the structure of the DNA-protein is stable by the presence of RecA protein and without RecA protein it is impossible to form or produce a triple-stranded DNA.
Therefore, in the field of biogenetics or the like, if one requires or wishes to use a triple-strained DNA, there are many restrictions as long as the triple-stranded DNA must remain in a complex with RecA Thus, a need exists to develop a method of forming a RecA protein free triple-stranded DNA whose structure remains stable.
Accordingly in order to meet the above need, the present invention provides a triple-stranded DNA whose structure can remain stable even if no protein is contained in its complex, a method of forming such a triple stranded DNA, and Southern hybridization employing such a triple-stranded DNA.
A first aspect of the present invention is to provide a method for forming a three-stranded DNA which comprises the steps of:
(a) DNA-protein complex forming process for forming a DNA-protein complex, wherein (1) a linearized double-stranded DNA, (2) a linearized single-stranded DNA including a base sequence, the base sequence being substantially complementary to a base sequence which extends from a base near 5xe2x80x2-end of one of DNA chains of the double-stranded DNAs, (3) a recombinant protein which is at least one of a homologous protein and another protein which is similar thereto in function, and (4) a nuclease which is at least one of an Exonuclease I of Escherichia coli and another protein which is similar thereto in function are reacted in order that in the DNA-protein complex an end neighboring inclusion region including the 5xe2x80x2-end of one of the DNA-chains of the double-stranded DNAs is bound to a complementary region including the substantially complementary base sequence of the single-stranded DNA under a participation of at least the recombinant protein; and
(b) protein deactivating process deactivating both the recombinant protein and nuclease to bind the complementary region of the single-stranded DNA to the end neighboring inclusion region of the double-stranded DNA.
In accordance with the first aspect of the present invention in the DNA-protein complex forming process, the DNA-protein complex is formed from the double-stranded DNA, the recombinant protein, and the nuclease. Thereafter, in the subsequent protein deactivating process, deactivating the recombinant protein and the nuclease makes it possible to form the triple-stranded DNA which has the 3-chain forming region which is formed by the bonding between the end neighboring inclusion region of the double-stranded DNA and the complementary region of the single-stranded DNA. Thus-formed triple-stranded DNA can remain its structure i.e. cannot be disassociated, even if a heat is applied thereto more or less, without having to pare a specially prepared protein such as RecA for stabilizing the structure. It is to be noted that the present invention makes it possible to form the 3-chain forming region on both of the end neighboring inclusion regions of the double-stranded DNA other than the formation on one of the end neighboring inclusion regions.
The above-mentioned method for forming a triple-stranded DNA is applicable to, say, southern hybridization.
In the conventional southern hybridization, the operations, for example, are as follows. As a target DNA a restriction-enzymatically cleaved linearized double-stranded DNA is prepared, while as a probe DNA a single-stranded DNA is prepared whose 5xe2x80x2-end is labeled with 32P with usage of T4 Polynucleotide kinase and [xcex3-32P]. The target DNA (i.e. the double-stranded DNA) is subjected to agarose gel electrophoresis and the agarose gel is placed on a membrane for vacuum filtration or the like and the target DNA (i.e. the double-stranded DNA) in the agarose gel is transfer onto the membrane. Thereafter, the target DNA (i.e. the double-stranded DNA) is made into a single-stranded state by disassociation as well as the resulting target DNA (i.e. the double-stranded DNA) is made immobilized on the membrane. Then, the resulting membrane is immersed into a solution of the probe DNA (i.e. a solution of the labeled single-stranded DNA) for hybridization and the membrane is made cleaned. Thereafter, the membrane is taken with a picture of autoradiogram to record a signal on an X-ray film which results from the labeled probe, DNA (i.e., the labeled single-stranded DNA).
On the other hand, a southern hybridization which depends on the present invention can be performed, for example, according to the following steps. In detail, like the conventional southern hybridization, a target DNA (i.e. a double-stranded DNA) and a labeled probe DNA (i.e. a labeled single-stranded DNA) are prepared At this stage, if the present invention is employed to do southern hybridization, a DNA-protein complex is formed by reacting such DNAs, a recombinant protein, and a nuclease (DNA-protein complex forming process). Thereafter, the recombinant protein and the nuclease are made deactivated to form a stable triple-stranded DNA having a 3-chain forming region (Protein deactivating process). Next, the resulting triple-stranded DNA is subject to agarose gel electrophoresis. Thereafter, the agarose gel is placed on filter paper to dry with drying device. The resulting gel is taken with a picture of autoradiogram to record a signal on an X-ray film which results from the labeled probe DNA (i.e. the labeled single-stranded DNA).
Thus-performed southern hybridization according to the present invention can be of less time operation and less cumbersome, when compared to the conventional southern hybridization. The reason is that southern hybridization according to the present invention eliminates skilled and/or long-time required operations such as transfer of DNA in agarose gel on membrane, immersing such membrane into probe DNA solution, and membrane cleaning. It is to be noted that the above description can be applied when a single-stranded DNA is used which is labeled chemically with e.g. a fluorescent material or phosphors.
At this stage, so long as in the triple-stranded DNA the 3-chain forming region is formed by chemical bonding between one of the end neighboring inclusion regions of the double-stranded DNA and the complementary region of the single-stranded DNA, the mode of such the chemical bonding is out of concern. That is to say, it is not necessary to find, between the double-stranded DNA and the single-stranded DNA, a specific chemical bonding mode such as Watson-Click type base pair or Hoogstein type base sequence. It is enough to find any mutual reaction between the double-stranded DNA and the single-stranded DNA which results in formation of a triple-stranded DNA.
Any double-stranded DNA is available so long as it is linearized. That is to say, the base sequence is out of concern and its upper limit of the chain length is not limited. Thus, or example, a huge DNA having a 3000 Mbp is available which is similar to that of human gene. Of course, the derivation of the double-stranded DNA is out of question. Thus, it is possible to use, for example, the following DNAs:
DNA derived from each of virus microbe, plant, and animal genes;
DNA obtained by reforming the above-mentioned DNA,
plasmid DNA included in microbe;
chimeric DNA obtained by inserting a heterologous DNA fragment into plasmid DNA; and artificially synthesized oligonucleotide.
Any single-stranded DNA is available so long as it is a linearized DNA which includes a base sequence which is substantially complementary to a base sequence which begins at a near portion of the 5xe2x80x2-end of one of the DNA chains of the double-stranded DNA. That is to say, so long as this condition is satisfied, the base sequence of the single-stranded DNA is out of concern. Like the double-stranded DNA, wit respect to the single-stranded DNA no upper limit of the DNA chain exists in theory and the derivation is out of question.
The above-mentioned substantial complementary will be about 70-80% or above, preferably 100%. The reason is that as the substantial complementary becomes higher the stability of the 3-chain forming region (i.e. the triple-stranded DNA) also becomes higher. However, depending on the length of the complementary region, the degree of complementarily will vary. In addition, it may be sometimes impossible to put a case and another case in the same class, the former class being of an even complementary (e.g. 70%) throughout the complementary region, the latter being of uneven distribution of a higher complementary zone (e.g. 9%) a lower zone complementary zone (e.g. 40%).
The single-stranded DNAs include a contemporary region, resulting in the entire single-stranded DNA complexing with the double-stranded DNA or the single-stranded DNA may also include another region that does not complex with the double-stranded DNA. However, the former is preferred from viewpoint of making forming a more stable triple-stranded DNA. It is to be noted that the reason for including a base sequence which is substantially complementary to a base sequence which begins at a near portion of the end of one of the DNA chains of the double-stranded DNA is as follows: If the single-stranded DNA is complementary to only a portion (e.g. a central-positioned base sequence) other than the base sequence which begins at a near portion of the end of one of the DNA chains of the double-stranded DNA, indeed it is possible to form a stable DNA-protein complex in the DNA-protein complex forming process, however deactivating the protein in the protein deactivating process causes the double-stranded DNA and the single-stranded DNA to disassociate, thereby failing to form or produce a stable triple-stranded DNA.
Similarly, the reason for including a base sequence which is substantially complementary to a base sequence which begins at a near portion of the 5xe2x80x2-end of one of the DNA chains of the double-stranded DNA is as follows: If the single-stranded DNA is complementary to only a base sequence which begins at a near portion of the 3xe2x80x2-end of one of the DNA chains of the double-stranded DNA, indeed it is possible to form a stable DNA-protein complex in the DNA-protein complex forming process, however deactivating the protein in the protein deactivating process causes to disassociate the double-stranded DNA and the single-stranded DNA, thereby failing to form or produce a stable triple-stranded DNA.
Any recombinant protein is available so long as it is a homogeneous recombinant protein or an analog thereof(i.e. a substance whose function is similar to homogeneous recombinant protein) and enables the formation of stable complex of the triple-stranded DNAs. Examples include: RecA protein derived from Escherichia coli, Thermus thermophilus, Other multi-functional proteins coded by RecA gene in intestinal bacteria, RecA-like protein derived from one of Agrobacterium tumefaciens, Bacillus subtilis, Methylophilus methylotrophus, Vibrio cholerae, and Ustilago maydis. The RecA-like includes also Saccharomyces cerevisiae and human genes.
A reformed protein which is produced by reforming one of these proteins is available so long as the reformed protein has a function similar to that of the latter protein. An example of the reformed protein is one which is a gene product produced or derived from a gene encoding homogeneous recombinant protein by e.g. site directed mutagenesis, which includes an amino acid sequence in which one or more amino acids are made deficient, replaced, or added, and which is similar, in function, to the homogeneous recombinant protein. In addition, a protein fragment of RecA protein (i.e. RecA fragment) which is of similar function to the homogeneous recombinant protein may be used.
Any nuclease may be used and can be chosen from Exonuclease I, preferably obtained from Escherichia coli, or a protein which is functional similar thereto. As the latter, an Exonuclease-I-like protein is available which is derived from, for example, eucaryotic organism (eucaryotic plant and/or animal) and other Exonuclease-I-like proteins, which are derived from, for example, prokaryotic such as Bacillus. In addition, a reformed protein which is produced by reforming one of these proteins is available so long as the reformed protein has a function similar to that of the Exonuclease-I-like protein An example of the reformed protein is one which is a gene product produced or derived from an Exonuclease-I gene by, for example, site directed mutagenesis, which includes an amino acid sequence in which one or more amino acids are made deficient, replaced, or added, and which is similar, in function, to the Exonuclease-I. In addition, a protein fragment of Exonuclease I gene (i.e. Exonuclease-I fragment) can be used provided the function is of similar to the full-length Exonuclease I.
The DNA-protein complex forming process is preferred or desired to be performed in buffer in the presence of nucleotide triphosphate or its analogs for effective formation of a stable DNA-protein complex. The buffer can be altered, for making the reaction conditions best, depending on the to-be-used recombinant protein and nuclease. For example, a tris-family buffer may be used whose pH is adjusted to about 4.0-9.0, preferably about 7.0-8.0. In general, the buffer is set to be, in concentration, about 10-100 nM, preferably about 30 nM.
As the nucleotide triphosphate or its analog, the following can be used: adenosine 5xe2x80x2-triphosphate (ATP), guanosine 5xe2x80x2-triphosphate (GTP), UTP, CTP, adenosine (xcex3-thio)-triphosphate (ATP-xcex3 S), guanosine (xcex3-thio)- triphosphate (GTP-xcex3 S), dATP, dUTP, and dCPT. Each of these substances is also available in combination with a nucleo diphosphate such as ADP. It is to be noted that particularly in a system for forming a DNA-protein complex if the nucleotide triphosphate such as ATP is accompanied biochemical decomposition, using the analog such as ATP-xcex3 S of the nucleotide triphosphate is recommended or preferred. The consistency of the nucleotide triphosphate is set to be 0.1-10 nM, preferably about 5 mM.
The concentration of each of the nucleic acids (i.e. the double-stranded and single-stranded DNA) in the reaction solution can be altered so long as the former is dissolved in the latter. The ratio of the single-stranded DNA relative to the double-stranded DNA is preferred to be about 1-100 times in molar ratio.
Adding 1 molecule of the recombinant protein to 3 base sequences of the single-stranded DNA is preferable. However, it is to be noted that the optimal amount changes slightly depending on the recombinant protein per se to be added. In addition, adding about 1 unit nuclease into the double-stranded DNA per 1 xcexcg. The optimal ratio also varies more or less depending on the nuclease per se to be added. The resulting reaction solution makes it possible to form a DNA-protein complex by being held at a temperature of4-60xc2x0 C., preferably about 37xc2x0 C., for a time duration of 5 minutes or above, generally about 60 minutes.
Instead of the above-described method in which the reaction solution is added with all other substances and thereafter is held at a temperature for a time duration, the following can be employed. In detail, to begin with, the double-stranded DNA, single stranded DNA, and recombinant protein are added in the buffer which includes therein nucleotide triphosphate etc at a temperature of 4-60xc2x0 C., preferably about 15 37xc2x0 C., for a time duration of about 2-5 minutes or above, preferably about 10 minutes. The nuclease is added to the resulting reaction solution and is further held at a temperature of 4-60xc2x0 C., preferably about 37xc2x0 C., for a time duration of 5 minutes or above, generally about 30 minutes. These reaction make it possible to form a remarkable stable DNA-protein, which is likely because the Exonuclease I or the like is an enzyme which cleaves a single-stranded DNA from its end, an at-first addition of the Exonuclease I or the like into the reaction solution sometimes may delete the single-stranded DNA. However, the later addition of the Exonuclease I or the like as mentioned above makes it possible to establish an earlier or at-first bond of the recombinant protein with the single-stranded DNA, resulting in the single-stranded DNA protected or free from the Exonuclease I or the like. Thus, the single-stranded DNA is difficult to cleave, thereby making it possible to form a stable DNA-protein complex.
To deactivate the protein, one of or a combination of the following arm, added to the reaction solution: chelate agent (e.g. ethylenediaminetetraacetic acid), addition of sodium dodecyl sulfate (SDS), and starch degrading enzymes (e.g. proteinase K). Thereafter, the resulting reaction resolution is held at a temperature of about 37xc2x0 C. for a time duration of 10 minutes and the triple-stranded DNA can be recovered or isolated therefrom. Such recovery or isolation can be accomplished by column chromatography or by separating the DNA temporally using methanol precipitation.
In the method for forming a triple-stranded DNA, it is preferred that the triple-stranded DNA whose substantial complementary base sequence is a 20 mer or above.
In theory, even if the complementary region of the single-stranded DNA is short, it is possible for form a 3-chain forming region by being bound to the end neighboring inclusion region. However, when the complementary region of the single-stranded DNA is too short (e.g., 20 mer or less), the formed triple-stranded DNA is not stable. On the contrary, the present invention employs a single-stranded DNA whose substantial base sequence is about a 20 mer or above. In brief forming a more stable triple-stranded DNA can be made possible. It is to be noted that employing a single-stranded DNA whose substantial base sequence is of about 30 mer or above makes it possible to make the formed triple-stranded DNA more and more stable, which is preferable.
Moreover, in each of the above-described triple-stranded DNA forming methods, the single-stranded DNA is preferred to have a base sequence which is substantially complementary to a base sequence which begins within about 20 nucleotides from the 5xe2x80x2-end of one of the DNA-chains of the single-stranded DNA.
As described above, so long as a single-stranded DNA includes a base sequence which is substantially complementary to a base sequence which begins at the 5xe2x80x2-end of one of the DNA-chains of the single-stranded DNA, binding the substantial complementary region to the end neighboring inclusion region makes it possible to forma 3-chain forming region. However, as the site to which the complementary region is bound makes a distance longer from the end of the double-stranded DNA, the 3-chain forming region is made unstable and easy to disassociate. In other words, as the region of only double-stranded increases in length, the formation of the triple-stranded DNA becomes less an less stable due to the stress caused by the double-stranded region on the triple-stranded region.
On the contrary, the present invention employs a single-stranded DNA which has a base sequence which is substantially complementary to a base sequence which begins at a within about 20 nucleotides from the 5xe2x80x2-end of one of the DNA-chains of the single-stranded DNA. That is, the complementary region of the single-stranded DNA is complementary to a region which begins at the very near end of the double-stranded DNA. Thus, in the formed triple-stranded DNA according to this method, the 2-chain forming region appears as an extension of the 3xe2x80x2-end of the single-stranded DNA in smaller length or fails to form. Thus, the structure stress resulting from the formation of the 2-chain forming region becomes difficult to generate, thereby stabilizing the 3-chain forming region. In conclusion, the present invention makes it possible to form a more and more stable triple-stranded DNA.
In particular, the single-stranded DNA is preferred to include a base sequence which is complementary to a base sequence which begins at the 5xe2x80x2-end of one of the DNA chains of the double-stranded DNA. This results in a 2-chain forming region not being formed on an extension of the 3xe2x80x2-end of the single-stranded DNA Thus, the 3-chain forming region is made stable in maximum, which makes it possible to form a most stable triple-stranded DNA. It is preferred that the complementary region of the single-stranded DNA includes a base sequence of about 60 nucleotides or less.
In the above-described method for forming a triple-stranded DNA, the recombinant protein is preferably RecA protein of Escherichia coli and a reformed protein which is produced by reforming this RecA protein so as to have a similar function thereto. In view of commercially availability, safety, and functionality, the RecA protein derived from Escherichia coli is desirable. An example of a reformed protein is one which is a gene product produced or derived from a RecA gene by, e.g., site directed mutagenesis, and includes an amino acid sequence in which one or more amino acids are made deficient, replaced, or added, and which is similar, in function, to the RecA protein. A protein fragment is also available, which is a product of reforming RecA protein gene and which is of a function similar thereto. In addition, a fragment of RecA (i.e. a RecA fragment) is also available which is of a function similar to the RecA protein.
The present invention also provides a kit for forming a triple-stranded DNA is available which includes at least either of a homologous recombinant protein and a protein having a function similar to that of the homologous recombinant protein, at least either of an Exonuclease I of Escherichia coli and a protein having a function similar to that of the Exonuclease I, at least either of a nucleotide triphosphate and its analogy, and a buffer.
Using the above-mentioned kit forming a triple-stranded DNA makes it possible to form a DNA-protein complex easily by way of a bond of the double-stranded DNA, the single-stranded DNA, and the Exonuclease I which is reacted in the buffer in which the nucleotide triphosphate is added. The resulting DNA-protein complex makes it possible to for a stable triple-stranded DNA by deactivating the proteins (i.e. the homologous recombinant protein, Exonuclease I, or the like).
The present invention also provides A triple-stranded DNA is made up of a linearized double-stranded DNA and a linearized single-stranded DNA including a base sequence, the base sequence being substantially complementary to a base sequence which extends from a base near 5xe2x80x2-end of one of DNA chains of the double-stranded DNA, the linearized double-stranded DNA and the linearized single-stranded DNA forming a 3-chain forming region in such a manner that an end neighboring inclusion region includes the 5xe2x80x2-end of one of DNA-chain of the double-stranded DNA being bound to a complementary region including the substantially complementary base sequence of the single-stranded DNA.
Unlike the conventional triple-stranded DNA, the newly invented triple-stranded DNA does not include protein and is formed only by the bond or coupling between the double-stranded DNA and the single-stranded DNA. The complementary region of the single-stranded DNA includes the 3-chain forming region bound to one of the end neighboring inclusion regions of the double-stranded DNA. Thus formed triple-stranded DNA can maintain its structure in stable fashion, even if more-or-less heat is applied thereto, without having to include a specially prepared substance such as protein or RecA protein. It is to be noted that the triple-stranded DNAs of the present invention also include one in which each of the end neighboring regions of the double-stranded DNA is formed with the 3-chain forming region
The above-described triple-stranded DNA may be used in a southern hybridization protocol. The target DNA would be a linearized double-stranded DNA and is prepared by cleavage with a suitable restriction enzyme, while as a probe DNA the a single-stranded DNA is prepared whose 5xe2x80x2-end is labeled with 32p using T4 Polynucleotide Kinase and[xcex3-32P]ATP. These DNA molecules are used to form a triple-stranded DNA such that the triple-stranded DNA includes a 3-chain forming region which is in the form of a bond between the complementary region of the single-stranded DNA and at least one of end neighboring inclusion regions. The triple-stranded DNA is subjected to agarose gel electrophoresis and the resulting agarose gel is placed onto a filtering paper or the like to dry with a gel drier. Then, autoradiogram of the agarose gel is taken to record a signal resulted from the probe DNA (i.e. labeled single-stranded DNA) on an X-ray film.
Thus established southern hybridization utilizing the invented triple-stranded DNA requires no additional cumbersome steps often associated with Southern hybridization techniques, such as transfer of the-agarose-gel DNA onto a membrane, immersing the resulting membrane in a probe DNA solution, and cleaning the membrane, resulting in easy doing the newly established southern hybridization for a shorter time duration, when compared to the conventional southern hybridization. This can be seen when the probe DNA is in the form of a chemically labeled single-stranded DNA which is labeled with a fluorescent material uses or the like.
The 3-chain forming region can be formed on the end neighboring inclusion region of the double-stranded DNA. However, as the 3-chain forming region moves away from the end of the double-stranded DNA, the 3-chain forming region becomes unstable and disassociates easily. When the double-stranded DNA becomes longer and is formed on the extension of the 3xe2x80x2-end, the structure stress which results from the existence of this double-stranded DNA makes the 3-chain forming region unstable, whereby the 3-chain forming region becomes dissociates easily. Contrary to this, according to the present invention, no 2-chain forming region is formed on the extension of the 3xe2x80x2-end of the single-stranded DNA which constitutes the 3-chain forming region or the 2-chain forming region is as short as 20 basepairs or less even formation thereof Thus, according to the present invention the structure stress which results from the existence of the 2-chain forming region does not form thereby resulting in stabilized 3-chain forming region. It is to be noted that the 3-chain forming region is desired to have a base sequence of about 60 nucleotides or less per unit DNA chain.
In such Southern hybridization protocols to detect the presence of nucleic acid molecules, e.and, double-stranded DNA, the method will include the following steps; an electrophoresis process for subjecting a triple-stranded DNA to agarose gel electrophoresis, the triple-stranded DNA including a linearized double-stranded DNA; and a linearized single-stranded DNA including a base sequence, the base sequence being substantially complementary to a base sequence which extends from a base near 5xe2x80x2-end of one of DNA chains of the double-stranded DNA, the linearize double-stranded DNA and the linearized single-stranded DNA forming a 3-chain forming region in such a manner that an end neighboring inclusion region includes the 5xe2x80x2-end of one of DNA-chain of the double-stranded DNA being bound to a complementary region including the substantially complementary base sequence of the single-stranded DNA; a dry process for drying the agarose gel including the triple-stranded DNA; and a detection process for detecting a signal from the agarose gel which results from the labeled single-stranded DNA.
It is to be noted that labeling the single-stranded DNA can be made with either radioactive element or chemical substance such as fluorescence material. Labeling the single-stranded DNA with radioactive element makes it possible to increase the detection ability of the southern hybridization, while labeling the single-stranded DNA with chemical substance makes it possible to perform each of the processes in safety and makes it possible to automate each of the processes.